Crosslinked Hyaluronan Derivative, Method of Preparation Thereof, Hydrogel and Microfibers Based Thereon

Abstract

The invention relates to a crosslinked hyaluronan derivative in the form of a hydrogel or microfibers, and to a method of preparation thereof, consisting in a C—C coupling reaction. The C—C coupling reaction is performed via reaction of a hyaluronan derivative carrying a terminal aryl-halide and/or aryl-borate group, and a hyaluronan derivative carrying an alkenyl or alkynyl group, in water, phosphates buffer or a mixture of an organic acid and an alcohol, and in the presence of a palladium active catalyst. The palladium active catalyst may be e.g. a complex of palladium (II) acetate and an inorganic or organic base, or a complex of palladium (II) and 2-amino-4,6-dihydroxypyrimidine.

1 . A process of preparation of a crosslinked hyaluronan derivative, characterized in that it is carried out by a C—C coupling reaction in water, phosphates buffer or a mixture of an organic acid and an alcohol, and in the presence of a palladium active catalyst, wherein the C—C coupling reaction takes place between a hyaluronan derivative carrying a terminal aryl-halide and/or aryl-borate group, and a hyaluronan derivative carrying an alkenyl or alkynyl group. 
     
     
         2 . The process according to  claim 1 , characterized in that first a secondary amine hyaluronan derivative carrying a terminal aryl substitute, according to the formula (I), is prepared: 
       
         
           
           
               
               
           
         
         wherein X=halogen or a borate functional group, 
         then a secondary amine hyaluronan derivative carrying an unsaturated compound that contains a triple or a double bond, according to the formula (II) or (III), is prepared: 
       
       
         
           
           
               
               
           
         
         wherein R 1  is an aliphatic C 1-15  group, 
         and then the derivative of the formula (I) is mixed with the derivative of the formula (II) or (III), then a palladium active catalyst stable in water is added in order to obtain the crosslinked hyaluronan derivative. 
       
     
     
         3 . The process according to  claim 2 , characterized in that X is I, Br or B—(OH) 3 , and R 1  is methylene or ethylene. 
     
     
         4 . The process according to any of the preceding claims, characterized in that the palladium active catalyst is selected from the group comprising a complex of palladium (II) acetate and an inorganic or organic base, and a complex of palladium (II) and 2-amino-4,6-dihydroxypyrimidine, while the concentration of the palladium active catalyst in the reaction mixture is within the range from 1×10 −5  to 1×10 −3  M. 
     
     
         5 . The process according to  claim 4 , characterized in that the palladium active catalyst is a complex of palladium (II) acetate and an inorganic or organic base, the concentration of the palladium active catalyst in the reaction mixture is within the range from 1×10 −4  to 1×10 −3  M, preferably 5×10 −4 , while the resulting crosslinked hyaluronan derivative is in the form of a hydrogel. 
     
     
         6 . The process according to  claim 5 , characterized in that the base is selected from the group comprising DABCO, TEMED, TEA, secondary phosphates, such as K 2 HPO 4 , carbonates, such as CsCO 3 . 
     
     
         7 . The process according to  claim 5 , characterized in that the base is TEMED or DABCO. 
     
     
         8 . The process according to  claim 4 , characterized in that the derivative of the formula (I) is mixed with the derivative of the formula (II) or (III) and the mixture is extruded into a coagulation bath in order to obtain hyaluronan derivative microfibers, and then the microfibers are transferred to the crosslinking bath containing a palladium active catalyst in order to obtain crosslinked hyaluronan-derivative microfibers. 
     
     
         9 . The process according to  claim 8 , characterized in that the palladium active catalyst is a complex of palladium (II) and 2-amino-4,6-dihydroxypyrimidine and the concentration of the palladium active catalyst in the crosslinking bath is within the range from 1×10 −5  to 1×10 −4  M, preferably 5×10 −5 . 
     
     
         10 . The process according to  claim 8  or  9 , characterized in that the coagulation bath consists of a mixture of an alcohol and an organic acid and the crosslinking bath consists of a mixture of an alcohol and lactic acid and the palladium active catalyst. 
     
     
         11 . The process according to  claim 10 , characterized in that the alcohol in the crosslinking bath is selected from methanol, ethanol and isopropanol. 
     
     
         12 . The process according to any of the preceding claims, characterized in that the crosslinking is carried out at temperatures of between 25 and 100° C., preferably 60° C. 
     
     
         13 . A hydrogel according to the formula A or B 
       
         
           
           
               
               
           
         
         wherein R 1  is an aliphatic C 1-15  substituent. 
       
     
     
         14 . The hydrogel according to  claim 13 , characterized in that R 1  is methylene or ethylene. 
     
     
         15 . A microfiber based on a hyaluronan derivative according to the formula A or B 
       
         
           
           
               
               
           
         
         wherein R 1  is an aliphatic C 1-15  substituent. 
       
     
     
         16 . The microfiber according to  claim 15 , characterized in that it has the diameter of 100 to 300 μm.